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Full text of "A Study of Plastids and Mitochondria in Pressia and Corn"

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AMERICAN 
JOURNAL OF BOTANY 

Vol. VI June, 1919 No, 6 

A STUDY OF PLASTIDS AND MITOCHONDRIA IN PRESSIA 

AND CORN 

W. C. Twiss 

In this paper certain observations upon plastids and mitochondria are 
recorded. In my opinion it would be premature as yet to formulate any 
conclusions as to the fundamental significance of these bodies, though the 
preparations I have obtained are clean-cut and definite. 

In order to make clear the position assumed in regard to the structures 
in question, it may be well to state, at the outset, that the term plastid will 
be used to include not only the leuco-, chloro-, and chromoplasts, but also 
the Anlagen for the same. The name mitochondria, on the other hand, 
will be restricted to those granules which are not, in general, preserved by 
the usual methods of fixation — those which, in other words, are dissolved 
in acetic acid or in alcohol and are fixed by the use of osmic acid, formalin, 
etc. The mitochondria, moreover, color more or less specifically with 
various stains. I shall in general use Benda's term mitochondria, rather 
than others that have been proposed, though the etymology of the word 
implies a thread-like form not always present. 

In cells prepared by what are known as the mitochondrial methods, 
these bodies, by reason of their number and intense affinity for the dyes 
become in many cases quite the most striking features of the protoplast. 
The only reason that they were neglected by cytologists for so long a time is 
the fact that they are dissolved by the processes commonly used to demon- 
strate nuclear phenomena. 

Interest in the granular constituents of the cytoplasm has greatly in- 
creased in the last few years, though the idea of their importance is not a 
new one. To Altmann, in 1886, is due the formulation of what is generally 
known as the granular theory of protoplasmic structure. Hanstein, in 
1882, had maintained that protoplasm is made up of minute granules, which 
he termed microsomes, and a homogeneous fluid in which the microsomes 
float. Altmann, using a special technique, consisting essentially of fixation 
with osmic acid and potassium dichromate, was able to demonstrate the 
presence in various tissue cells, as well as in the chromosomes, of numerous 
granules to which he gave the name of bioblasts. These bodies he regarded 
as possessing an independent existence, and to them he imputed the power 
of growth and of multiplication by division. He also believed that they 
[The Journal for May (6: 181-216) was issued June 20, 1919.] 
217 



218 W. C. TWISS 

are transformed into the products of secretion such as fats, glycogen, pig- 
ments, etc., in the cells. But Altmann, besides claiming for his bioplasts 
the powers already noted, which are to a certain extent the same as those 
believed by modern exponents of the mitochondrial theory to reside in the 
mitochondria, held that the bioplasts are the morphological units of living 
matter, constituting the essential elements of protoplasm. The difference, 
in short, between Altmann's hypothesis and the more modern theories of 
the mitochondria lies in the fact that the bioplasts were postulated to possess 
an independence and autonomy, to quote Regaud, which the mitochondria 
as cell organs are not thought to exhibit. 

Benda, in 1898, having devised a more specific and definite method of 
fixation and staining, may be regarded as the founder of the modern mito- 
chondrial theory. He introduced the terms mitochondria and Chondrio- 
miten, from /uros a thread, and x^ptov a grain. Chondriosome and 
chondriocont, later introduced by Meves, have also come into use, the former 
being used synonymously with mitochondria, and the latter being applied 
by Meves to homogeneous threads. The collective term chondriome is also 
often employed. 

Regaud ('n) suggests the possibility that the mitochondria "fix" and 
"elaborate" the substances necessary for the functioning of the muscle 
cells, such as glycogen, and that they also perform a similar function in the 
case of the gland cells, such as the secretory cells of the kidney and of the 
salivary glands. He says: "Les mitochondries sont les organites sur lesquels 
se fixent les substances destinies au fonctionnement chemique de la cellule; ces 
organites concentrent les substances fixSes, les Slaborent et les transforment en 
produits de sScrStion, auxquels Us servent meme de supports, dont Us sont les 
plastes." The mitochondria are "les agents de V intussusception elective, 
c'est-a-dire, de V introduction dans la cellule des substances amen&es par le 
sang," 

Dubreuil ('13) believes that the mitochondria are responsible for the 
production of fat in the cells, through a process of differentiation. Accord- 
ing to Dubreuil, a lipoid vesicle is first formed, this process being followed 
by the development of a fat droplet. The diagrams to illustrate this 
process, showing the mitochondria becoming vesicular and forming both 
hollow spheres and "hand-mirror-like" forms, exhibit a remarkable simi- 
larity to the series of changes which Guilliermond and others show in their 
illustrations of the production of plastids by the mitochondria in plant cells. 

Van der Stricht ( '09) has presented some very suggestive studies upon 
the genesis of yolk-spheres in the egg. He finds that the mitochondria are, 
at first, confined to a region around the nucleus from which they migrate 
outward and are gradually transformed into yolk-spheres. 

Lewis and Lewis ('15) have employed a novel method for the study of 
mitochondria with most interesting and suggestive results. Portions of the 
living embryo of the chick were segregated under aseptic conditions and 
cultivated in Locke's solution, in hanging drop cultures. In such cultures 



A STUDY OF PLASTIDS AND MITOCHONDRIA 219 

the mitochondria may be studied in the living, growing condition, fixed 
while under observation and thus preserved as permanent preparations. 
Experimental methods may be employed, by which the effect of different 
stains and reagents may be observed directly. Such preparations, placed 
in the electric incubator at a temperature of 39 to 40 C, show a beginning 
of growth in from 10 to 20 hours. The new growth is attached to the cover 
glass and is several cell layers in thickness at first, thinning out to a single 
cell layer around the edges. Although conditions are necessarily somewhat 
different from those of the normal somatic environment, especially as regards 
circulation, and in the limited supply of oxygen since the cultures must be 
hermetically sealed, the processes of growth and of mitosis seem to go on as 
usual for a period of about three days. After this the process slows down, 
growth ceases, and the cells finally die. 

Mitochondria, conforming to the usual criteria, were found to be present 
in all cells studied. Osmic acid vapor proved to be the best fixative, while 
the vapors of acetic and other acids caused immediate and total disintegra- 
tion of the mitochondria. The bodies appeared as threads and granules 
of the most varied shapes and sizes, just as they have so often been described 
in fixed preparations. They are not, however, constant in form or in size 
under these conditions, but are constantly changing in appearance. They 
are described as undergoing division, as fusing to form larger bodies, and 
as disappearing and reappearing in the cells in a manner not accounted for. 
During mitosis they are distributed regularly about the cell, so that they 
are apportioned to the daughter cells in approximately equal numbers. 
No connection of the mitochondria with the production of fats, such as 
described by Dubreuil ('13), was noted in these preparations. In conclu- 
sion, the authors state: "The mitochondria are extremely variable bodies, 
which are continually moving and changing shape in the cytoplasm. They 
appear to arise in the cytoplasm and to be used up by cellular activity. 
They are, in all probability, bodies connected with the metabolic activity 
of the cell." 

Lewis and Robertson ('16) found that the above described method of 
tissue culture was well adapted to the study of spermatogenesis in the 
grasshopper, Chorthippus curtipennis. In the young spermatid the mito- 
chondria were in the form of a granular Nebenkern. After certain internal 
changes, the import of which was not clear, the Nebenkern was seen to divide 
into half-spheres. These half-spheres then elongated to form granular sacs, 
which, as the tail grew out, formed two irregular strands. These irregular 
strands finally fused to form "two continuous threads of even width, ex- 
tending from the centrosome body, or middle piece, almost to the end of the 
tail." 

The authors conclude that "it does not seem possible that bodies which 
have to do only with the metabolic activities of the cell should undergo such 
an exact behavior as shown, for instance, by the division of the Nebenketn 



220 W. C. TWISS 

into two equal parts and the development of these two sacs of mitochondria 
into two long threads of mitochondria in the spermatozoon." 

There has been a tendency, especially among the earlier observers of 
mitochondria in plant cells, to ascribe to these bodies a nuclear origin. 
A perusal of the literature clearly indicates that the work of Goldschmidt 
( '04) is largely responsible for this, though the inception of the chromidial 
hypothesis doubtless owes its origin, as Dobell ('09) states, to the work of 
R. Hertwig, supplemented by that of Schaudinn, dealing with the occurrence 
of such bodies in the protozoa. 

Meves ('04), working with the tapetal cells in the anthers of Nymphaea 
alba, is credited with having made the first observations of mitochondria 
in plant cells. Meves shows two very clear-cut and beautifully drawn 
figures, of which his description is as follows: " Enthalt sie lange, unregel- 
massig gewundene, ziemlich dicke Faden, welche sich mil Eisenhamatoxylin 
intensiv schwarz gejarbt haben. Diese Faden konnen nicht wohl etwas anderes 
sein, als die von tierischen Zellen bekannten Chondromiten." 

It is not so much the question of origin, whether sui generis or chromidial, 
however, that has engaged the attention of later workers upon plant mito- 
chondria, as that of their relation to other structures in the cell and of their 
universality. Lewitsky and Pensa, working independently, have advanced 
a contention which promises to furnish material for controversy for some 
time to come. 

Lewitsky ('io) studies the root-tip and stem-tip of Asparagus officinalis 
treated according to the Benda method and stained with both the Benda 
stain and haematoxylin. He finds mitochondria corresponding to those 
described in animal cells, both in general appearance and in staining reac- 
tion, with no evidence whatever of a nuclear origin. The mitochondria 
appear short and rod-shaped, in the dermatogen; somewhat larger and with 
a tendency to swell at the ends, in the second layer; still larger in the third 
layer, while deeper in the assimilative tissue "dumb-bell" forms are seen, 
"similar to those well known in division figures of the chloroplasts." 
Next, still larger bodies are shown which appear as if they have come from 
the separation of the two halves of the "dumb-bells." These are followed 
by figures of the young chloroplasts, and finally by the mature bodies. 
Meanwhile, the earlier forms of the mitochondria, combined with the 
"division figures" of the intermediate regions, lead him to conclude that 
the mitochondria are the Anlagen of the chloroplasts. Upon fixing some of 
the same material in alcohol and acetic acid, Lewitsky found that the mito- 
chondria were no longer to be seen in the cells, while the chloroplasts ap- 
peared as usual. This is taken as evidence of a chemical as well as a mor- 
phological transformation of the mitochondria, in producing the chloro- 
plasts. 

Guilliermond ('n, '13, '14) has published a number of papers in which 
he describes the mitochondria in all sorts of plants, his purpose being, on 



A STUDY OF PLASTIDS AND MITOCHONDRIA 221 

the whole, not so much to demonstrate the presence of mitochondria therein 
as to substantiate his theory of their functional r61e. In addition to 
portraying the same processes of development as those described by his 
predecessors, Guilliermond seeks to show that the mitochondria of plant 
cells possess the same "elaborative" functions that have been postulated for 
animal mitochondria by Regaud and others. In a resume of the work upon 
mitochondria, published in "La Revue generate de Botanique" in 1914, he 
says: " Ces recherches demontrent surabondammentque les mitochondries sont 
desplastes,c'est-d,-dire des organites quielaborentl&s produits de secrition. . . . 
A la suite de ces recherches, la cellule apparait dSsormais avec un nouvel ele- 
ment: le chondriome, dont la presence est aussi constante et joue un rdle aussi 
essentiel que le noyau. . . . La decouverte des mitochondries transforme done 
la cytologies Mottier ('18) has discussed the literature on the relations of 
chondriosomes and plastids and it need not be further summarized here. 

Observations 

Although I was particularly concerned, in my own investigations, with 
obtaining evidence as to the relationship between mitochondria and plastids, 
the mitochondrial methods of fixing and staining were tested first upon 
animal tissue. A number of preparations were made from the testes of the 
grasshopper, Caloptenus femurrubrum. They were fixed with Benda's 
solution, with Bensley's,and with Flemming's strong solution, and stained 
in various ways, in order to compare the results obtained with different 
combinations. 

Benda's method of fixing and staining gave by far the best results, the 
mitochondria being well differentiated and shown in the characteristic 
changes through which they pass, as described by Lewis and Robertson 
('16) in their observations upon the behavior of the mitochondria during 
spermatogenesis in the grasshopper, Chorthippus curtipennis. In my 
preparations, Bensley's method gave unsatisfactory results in general 
protoplasmic differentiation, though the mitochondria were well preserved 
and stained. 

Benda's process, according to the formula given below, was therefore 
used in most of my plant material. 

Fixation. I. Benda's Flemming, 8 days. 

(1 percent chromic acid, 15 cc, 

2 percent osmic acid, 4 cc, 

3 drops acetic acid.) 
II. Wash in water, 1 hr. 

III. Pyroligneous acid (rectified) and chromic acid 1 per- 

cent, equal parts, 24 hrs. 

IV. Bichromate of potassium, 2 percent, 24 hrs. 
V. Wash in water, 24 hrs. 

VI. Dehydrate and imbed. Cut 5 microns thick. 



222 W. C. TWISS 

Staining. VII. Iron alum, 4 percent, 24 hrs. 
VIII. Rinse in distilled water. 
IX. Alizarine sodium sulphonate, 24 hrs. 

(1 to several cc, saturated alcoholic solution, in 80 to 
100 cc. distilled water.) 
X. Heat in crystal violet, 3 to 5 min. after vapor rises. 
(3 percent alcoholic solution crystal violet and aniline 
water, equal parts.) 
XI. Rinse in distilled water. 
XII. Destain in 15 percent acetic acid. 

XIII. Wash in running water, 5 to 10 min. 

XIV. Dry with filter paper. Dip in absolute alcohol. 
XV. Bergamot oil, followed by xylol. 

This stain gave a very beautiful result, the mitochondria being colored 
a dark violet or blue, with a background of old rose. Only in exceptional 
cases was the background too light to be satisfactory. 

Regaud's method of fixing and staining was also employed, to some 
extent, upon the plant tissues. 

Fixation. I. Bichromate of potassium, 3 percent, 80 vols., and com- 
mercial formalin, 20 vols., four days. 
II. Bichromate of potassium, 3 percent, eight days. 

III. Wash in water, 12 hrs. 

IV. Dehydrate, imbed, and cut 5 microns thick. 
Staining. V. Stain with iron-alum-haematoxylin (Heidenhain's 

method) . 

This is the formula as given by Guilliermond, who has used it in much 
of his work. In my preparations it gave good results, at times, while again 
the results might be very bad, possibly due to impurities in the formalin 
The Benda fixation does just as well as a preparation for the iron-alum- 
haematoxylin as for the crystal violet-alizarin stain, this combination being 
often used. 

Corn 

In the root-tips of corn, of the " Canadian Early, Yellow Flint" variety, 
fixed according to Regaud's formalin-bichromate method, the time being 
shortened to four hours in the fixative (I), and eight hours in the bichromate 
(II), the cytoplasm in the embryonic region appears gray and filled with 
exceedingly numerous jet-black mitochondria, when stained with iron- 
alum-haematoxylin. In this region the mitochondria are globular, ellip- 
soid, or short rod-shaped. In the root-cap, next to the tip, they are similar, 
gradually lengthening from short rod-shaped to elongated, filamentous 
forms as one passes from the embryonic region toward the periphery of the 
cap. 

In the root-tip proper, passing back into the region of elongated cells in 



A STUDY OF PLASTIDS AND MITOCHONDRIA 223 

the plerome, a marked change occurs. The mitochondria now appear as 
elongated, thread-like bodies, seeming to have arisen from the spherical 
and ovoid forms by a process of lengthening and thinning. Of these elon- 
gated forms, many appear hooked, or in some cases vacuolate, at the ends. 
Mingled with these thread-like structures are others which appear circular, 
as if a thread had formed a ring, or possibly a globular form had changed in 
appearance so that it resembles a hollow sphere. At times, chains of small 
granules are seen, probably due to the breaking up of a filament. Figure I 
shows the large number and greatly varied shapes of the mitochondria in 
this region. 

Passing outward from the region of elongated cells in the plerome and 
entering the periblem, the cells are found crowded with mitochondria, 
mostly spherical in shape. It appears as if the mitochondria do not, in 
general, elongate in this region, though they increase in size, approximately 
to the same extent in all directions. As will be shown later, these enlarged 
bodies of the periblem are in reality not mitochondria, but plastids, though 
they stain in exactly the same manner with the haematoxylin. Whether 
there are any plastids present in the plerome region as well, I am not as yet 
prepared to say. Figure 2 shows a cell from the periblem in mitosis, drawn 
to the same scale as figure 1, namely, nine hundred diameters. Figure 3 
shows a similar cell enlarged to twice the size. 

As may be seen from these drawings, the mitotic figure is shown very 
much as it appears in the usual method of fixation, with the exceptions to 
be noted. The spindle fibers are but faintly shown, if visible at all, although 
the general outline of the spindle appears as it normally does. The chro- 
mosomes are rather attenuated, though they sometimes show a shadowy 
outline of surrounding material, as if only the central part of their structure 
had been stained. The difference between this method of fixation and those 
methods designed primarily for showing nuclear structure, is much more 
marked in the resting cell. Here the nucleus appears, in general, more 
uniformly granular and less reticulate than it does in preparations fixed, 
say, in Flemming's strong fluid. The nucleole also is different, appearing 
much larger than we are accustomed to see it in fixed material. In short, 
the mitochondrial methods of fixation do not seem to alter the appearance 
of the protoplast so much as do the usual types of fixation, since with the 
mitchondrial methods the structure appears very much as it is described by 
Lewis and Lewis ('15) and by Lewis and Robertson ('16), in their obser- 
vations upon living tissue cells. 

The root-tips from which the above described preparations were made 
were grown in the laboratory during the coldest part of the winter and not 
under constant temperature conditions. To this I attribute the difference 
between the granular content of the cytoplasm in this material and in the 
preparations next to be described. 

Root-tips of the same variety of corn, grown later in the season and under 



224 W. C. TWISS 

more favorable conditions of temperature and light, prepared and stained 
by the Benda method, show a much greater proportion of the enlarged 
vesicular bodies which stain like mitochondria. In these preparations, 
however, there is very good evidence that these bodies are in reality plastids, 
since they show a lighter colored internal portion, evidently consisting of 
starch. In regions apart from the meristem these bodies are very abundant. 
In the plerome (fig. 4) the filamentous mitochondria often appear swollen 
at the ends or sometimes in other portions, while the plastids, ovoid or 
spherical in shape, may contain from one to several starch grains each as 
shown in figure 5. 

In the periblem, on the other hand, there are in the intermediate regions 
of the tip, ovoid, spherical, or irregular masses of a much more solid appear- 
ance, in general, but often showing a number of discrete spherical granules 
in their interior. Numerous smaller spherical or ovoid bodies are also 
present, scattered about through the cytoplasm, which are dark blue in 
color, taking the stain exactly as do the plastids and the filamentous mito- 
chondria. 

These preparations, stained and fixed according to the Benda method,- 
as previously stated, show the nucleus finely granular in appearance and 
of an old-rose color, as dark, relatively, as shown in the drawings, figures 
4 and 5. The nucleole is apparently of a denser consistency and stains a 
darker shade of the same color. The cytoplasm is very well preserved and 
stains somewhat lighter than the nuclear material. 

A slide of the above described material was freed from paraffin with 
xylol, bleached for a few minutes in hydrogen peroxide, and treated with 
potassium iodide-iodine solution, with the object of testing for starch. 
Under this treatment the bodies which have been referred to as plastids 
show a bluish color in their interior, but hardly pronounced enough to be 
considered a convincing demonstration of the presence of starch. A sub- 
sequent treatment of the slide with a solution of iodine in chloral hydrate, 
however, gave better results, differentiating the bodies so that they appear 
as plastids with included starch grains, as indicated by the blue color of the 
interior. 

Next, a similar slide from the Benda fixation was stained with the 
Flemming three-color process, the red being left decidedly strong. The 
mitochondria, both granular and filamentous, are now strongly stained by 
the safranin, while the plastids are colored blue. In the intermediate regions 
of the tip, in the periblem area, the plastids are rather lightly stained, and 
within each of them there are a varying number of spherical granules which 
are stained by the safranin in the same manner as the mitochondria, except 
that they are, generally, a brighter red. In figure 6, a, b, c, d, and e, a 
number of these plastids are shown with their included red-staining granules. 
As one leaves the intermediate regions of the tip and proceeds toward the 
proximal portion, the red bodies within the plastids gradually lose their 



A STUDY OF PLASTIDS AND MITOCHONDRIA 225 

sharply defined appearance and bright color, becoming gradually dimmer. 
Still farther back in the tip the red color disappears entirely, the plastids 
appearing more or less opaque or more uniformly blue in color, as shown in 
figure 6, g, h, and i. 

It has not been possible to determine, so far, how this association of the 
bright red granules with the light blue plastids comes about, though it might 
be imagined, from observations I have made, such as the appearance of 
two darkly staining red granules at the ends of a light blue ellipsoid, that 
the mitochondria are surrounded by, or become surrounded by, a substance 
which makes up the body of the plastid ; that they divide within this plastid 
substance and afterwards produce the starch grains within it, or themselves 
become changed into starch. This appearance suggests a relation to starch 
formation similar to that of the pyrenoid, as described by McAllister ('14). 

In other cases, however, bodies were observed which are made up of a 
dark red peripheral layer surrounding a light blue center. Both the latter 
structures and the ellipsoids occur in the intermediate regions of the peri- 
blem, between the red-staining mitochondria and the blue plastids with 
their red, granular inclusions. I wish to emphasize the fact that both the 
mitochondria and the plastids are exceedingly numerous in the regions 
indicated and that the staining reactions and the differentiation of the bodies 
described as occurring in the plastids are very definite. In many cells of 
the periblem, in the intermediate region of the tip, the nucleus is practically 
surrounded by a number of large plastids containing red-staining granules* 
while the cells nearer the tip are crowded with mitochondria which also 
color strongly with the safranin. Nevertheless, while the existence of 
these bodies is clearly demonstrable, I do not wish to imply that the seriation 
to prove their inter-relations is equally evident. 

Preissia commutata 

The situation in connection with the cytoplasmic inclusions of the 
liverworts and of the Bryophyta in general, appears to be in special need of 
investigation, not only on account of the fact that a perusal of the literature 
shows a considerable difference of opinion as to the real nature of the various 
bodies in question, but also on account of the very great interest attached to 
the group by virtue of its intermediate position in relation to the flowering 
plants on the one hand, and to the algae on the other. 

While the interest centers mainly in the plastids and their genetic 
relations, the oil bodies of the liverworts have received considerable atten- 
tion from investigators, with no very definite results as far as their real 
nature and origin is concerned. Pfeffer C74) is credited with having made 
the first really fundamental and comprehensive study of the oil bodies. 
In his opinion they are formed by the aggregation of a large number of 
small oil droplets which are already visible in the very young cells. While 
he, at first, maintained that the bodies originate in the cell sap, he later 



226 W. C. TWISS 

agreed that it might be possible that they come from the cytoplasm, but 
that they finally lie in the vacuole. He believed the principal constituent 
of the bodies to be a fatty oil, since their contents dissolve in alcohol, 
benzol, ether, etc. In addition to the fatty oil, some other material was 
found to be present, appearing as a residue after the solution of the oil. 
The membrane which he observed surrounding the bodies after they had 
been stained with iodine and with cochineal, was apparently composed of 
some protein material, insoluble in dilute acid and in alkalies. Since the 
bodies were unchanged after a three-months' cultivation of the plants in 
darkness, and since they were still present, in such cases, in the very young 
cells, just as in the plants which had grown in the light, he concluded that 
they have no significance in nutrition and that they are merely products of 
excretion. 

Wakker ('88) included the oil bodies of the liverworts under the "elaio- 
plasts," as he had named the oil-producing bodies which he had demon- 
strated in many phanerogams. Although these bodies appear, in life, to 
lie in the cell sap, Wakker showed by abnormal plasmolyosis that they, in 
reality, lie in the cytoplasm. He believed them analogous to leucoplasts 
and chloroplasts, holding that they multiply by division and are distributed 
to the daughter cells in mitosis. 

Von Kiister ( '94) believes that the oil bodies are formed from a protein 
"stroma" and that the apparent membrane seen in fixed material is an 
artefact. Since he was not able to see the membrane in living material, 
even with the strongest magnification, he considered it a precipitation 
membrane, formed by the interaction of the oil and the substance of the 
stroma. He showed that the membranes were not visible in material which 
had been fixed in osmic acid and stained with gentian violet. He also 
showed that a double membrane could be formed by the use of dilute 
alcohol, followed by strong. In regard to the nature of the bodies, he 
believed with Pfeffer that they are excretion products. He did not believe 
that the oil bodies undergo division and are handed down from cell to cell, 
but thought that they are newly formed in each cell. 

Garjeanne ('03) believes that it is possible to show that the oil bodies 
are in reality merely vacuoles filled with oil which is secreted from their 
walls; that they lie in a half-fluid transition substance; that they increase 
by division, and that the membrane is an artefact. He admits, however, 
that the picric acid which he used in his demonstrations acts very rapidly, 
so that observations upon young cells must be made within one minute 
after the application of the acid, before disorganization of the cell contents 
sets in. He compares the oil bodies to the leucoplasts in their origin from 
Anlagen, which he believes to be vacuoles in the case of the leucoplasts also. 
After being fully formed, the oil bodies, he says, are no longer capable of 
division, remaining thereafter unchanged. In addition to the vacuoles, or 
Anlagen, of the oil bodies, he describes other minute structures which are 



A STUDY OF PLASTIDS AND MITOCHONDRIA 227 

similar to the young stages of the oil bodies but which differ from them in 
their chemical properties. Since, in addition to the Anlagen of the oil 
bodies and to these structures which are similar to them, Garjeanne men- 
tions also the Anlagen of the chloroplasts, it would seem that he believes 
that there are present in the cells of some liverworts, at least three varieties 
of specifically different granules. 

Rivett ('18), in an account of certain observations upon Alicularia 
scalaris, finds that the results of staining or fixing the entire leaves with 
2 percent osmic acid "confirm the view that the oil is secreted in vacuoles." 
This author also finds certain refractive granules present in the cells of both 
the growing point and the older leaves, which differ in their chemical reac- 
tions from the young oil bodies, apparently agreeing with the observations 
of Garjeanne in this respect. In the meristematic regions a "chondriome- 
like structure" was observed, but no evidence was found "that the chon- 
driosomes were either transformed directly into plastids by a secretion 
within their own substance, or that they are the instigators of secretory 
action on the part of the protoplasm." No evidence was found that the 
refractive granules were chondriosomes, "since their appearance in the 
stained mature cells is quite different from that of the chondriome of the 
actively dividing cells." 

As already noted, mitochondria have also been described in the liver- 
worts by Scherrer ('13) and by Mottier, ('18), neither of whom was able 
to find any connection between these bodies and the chloroplasts. Scherrer 
made a special study of Anthoceros, a form which possesses the greatest 
interest since it has a "pyrenoid' in some of its chloroplasts, suggesting a 
close relationship with the algae in respect to its method of starch formation. 

The pyrenoid of Anthoceros has been described by McAllister ('14), 
who finds that it consists of from 20 to 300 minute lenticular bodies, which 
lie near the center of the chloroplast and which stain bright red with safranin. 
McAllister states that there can be no doubt that these bodies are trans- 
formed directly into starch, since "there is a gradual change of the color 
reaction, from the brilliant red of the pyrenoid bodies to the blue of the 
starch grains." On the other hand, he says that, in the cells of the arches- 
porium, in the spore mother cells, and in the assimilative cells of the sporo- 
phyte, starch is formed without the intermediary action of a pyrenoid — 
apparently arising de novo in the chloroplasts. 

The observations of Davis ('99) agree with those of McAllister in this 
respect, since be states that the first clear indication of the chloroplast in 
the spore mother cells of Anthoceros is the sharp staining of the starch 
grains — purple with the gentian violet. 

McAllister states, further, that there is no doubt that if the Anlagen of 
these bodies (the starch grains of the spore mother cells) are present in the 
plastids of the archesporial cells, they are too minute to be distinguished with 
the highest magnifications. This, however, does not necessarily follow, 



228 W. C. TWISS 

since neither McAllister nor Davis reports having tried the mitochondrial 
methods of fixation. 

Although Anthoceros, in general, has but one chloroplast in each cell, 
Campbell ('o6) has described a species from Jamaica which has several 
chloroplast^ — as many as eight in the cells of the inner tissue — so that the 
connection of Anthoceros with other liverworts, in this respect, is not so 
remote as might at first appear. All in all, Anthoceros is obviously a most 
interesting form and one upon which considerably more work is necessary. 

Observations 

Portions of the thallus of Preissia commutata, upon which the gamete- 
bearing discs were beginning to appear, were fixed according to Benda's 
formula, imbedded, and cut 5 microns in thickness. The smallest disc 
studied was about one millimeter in diameter. Figure 7 represents a portion 
of such a disc, as it appeared when stained with the Benda method, and 
with a magnification of three hundred eighty-four diameters. The darker 
cells in this section, three of which are shown in figure 7, present the same 
relatively dark appearance in the unstained, unbleached preparations. 
They are filled with a dense mass of thin-walled, spherical bodies which 
stain darkly with osmic acid as well as with the mitochondrial stains. 
Treatment with a preparation of alcannin shows the periphery of these 
cells made up of an alveolar substance, staining purplish gray, while the 
central portion contains a mass of material which stains a dark red. These 
central masses are the "oil bodies" of Pfeffer and others, or the "elaioplasts" 
of Wakker. 

In this section there are also differentiated two other sorts of bodies. 
The smaller, more uniform variety, which may be seen occupying the periph- 
ery of the cells, is apparently of a fatty nature, since they are somewhat 
darkened in the unbleached cells. They appear granular and plastic, 
being flattened more or less along the cell walls. There appears to be no 
difference between the periphery and the interior of these bodies, since no 
bounding membrane nor any lighter-colored area in the interior can be 
made out in the stained slides. From the larger ones, two microns or more 
in diameter, of which there are usually a larger number of about the same 
dimensions in these cells, they seem to grade down to extremely minute 
granules. 

The other bodies vary much more in size, there being no two of any one 
size in the cell. They include, doubtless, the bodies described as oil drop- 
lets by Pfeffer. Figures 8, 9, and 10 show a few of the cells taken from the 
same group as figure 7, but more highly magnified. The more uniformly 
colored bodies in these cells, mostly seen in side view around the periphery 
of the cell, belong to the first class, while the more rounded ones, with dark 
borders, belong to the second class. Of the latter, the larger ones may ap- 
pear to be in a state of division or fusion, a number, of varying sizes, often 



A STUDY OF PLASTIDS AND MITOCHONDRIA 229 

appearing in groups; figure 11, plate II, shows a peripheral cell from a 
young disc containing the two sorts of bodies of the second class. 

Returning to figure 7, as one proceeds from the periphery of the young 
disc toward its center, the bodies of the second class appear, on the whole, 
smaller. There seems to be a gradual decrease in their size correlated with 
an increase in their number, up to a depth of about two cell layers below 
the areolae. Here, bodies of this class begin to decrease in number, indi- 
cating a diminution in the amount of oil in the cells, while bodies containing 
from one to several starch grains begin to be seen: figures 8 and 9. 

This seriation, suggesting that at least some of the bodies are plastids, 
is better shown in figures 13 and 14, taken together, which were drawn 
from a somewhat older disc than that from which figure 7 was taken. 
Figure 14 shows two cells from still deeper within the disc, as compared with 
figure 13, from a central lenticular area in which all the cells are of this 
type and packed with storage starch. These plastids, for such they appear 
to be, are stuffed with starch and now appear merely as enveloping films, 
enclosing and separating the starch grains. The character of these starch 
grains, which show a definite hilum when stained in certain ways, as well as 
their general appearance and distribution, would indicate that they are, as 
already suggested, storage starch and that the grains are very likely strati- 
fied. It was also found on staining such a section as that shown in figure 12 
with iodine, that the starch reaction was given by all the bodies of this 
character, even to some of the smaller ones in the peripheral layer of the 
disc. Figure 15 represents a series of stages in outline as they are seen in 
the development of starch in this disc. 

In the chloroplasts of the thallus, starch is also present in large quan- 
tities, and toward the interior of the thallus there is a region in which the 
cells are moderately full of swollen plastids, each containing a number of 
plump starch grains, not at all lenticular in appearance as they are so often 
figured. Lenticular-shaped starch grains are found, however, in the chloro- 
plasts of cells at and near the periphery, especially in the vicinity of the 
growing point of the thallus. In the cells of the disc which contain starch, 
as well as in those of the thallus, the smaller, more plastic, and darker- 
staining bodies are still seen, arranged around the periphery. The largest 
of these should be the young chloroplasts. 

In the apical cell of the thallus and in its immediate vicinity, bodies may 
be seen which I take to be the same as those already described from the 
peripheral cells of the young disc, though they are much smaller in size. 
In this region, also, and especially in the filamentous growths therefrom, 
mitochondria of various shapes appear, very much as described by Mottier 
('18) for Marchantia. 

Discussion 

It was at one time the accepted belief among botanists that chloroplasts 
arise de novo in the cytoplasm. This is definitely stated to be the case by 



230 W. C. TWISS 

Sachs, in his Text-book of Botany, in 1882, where the process is compared 
to that of so-called free-cell formation. Since the work of Schimper, 
afterwards confirmed by that of A. Meyer, the opinion has become general 
that the three kinds of plastids found in plant cells, namely, leuco-, chloro-, 
and chromoplasts, are derived from minute, undifferentiated plastids which 
are sui generis structures of the cytoplasm. These chromatophores, as 
they are often called, were described, when seen in the living cell, as small, 
colorless, highly refractive bodies, recognizable in the egg and also in the 
embryonic cells. In older cells they have been said to retain the same 
appearance in some cases, while in others they become differentiated into 
leuco-, chloro-, and chromoplasts. 

Schimper and Meyer believed that the undifferentiated plastids multiply 
by division and are handed down from generation to generation — that they 
have an individual existence in the cells. Considerable difficulty, however, 
was encountered by them in their attempts to demonstrate the presence of 
the plastids in the egg } owing to the fact that they were not easily seen in 
the living cells, and, as was admitted, they were difficult to stain at that 
stage. As Guilliermond expresses it, "that part of their theory remained 
very hypothetical.' ' 

When the mitochondria were demonstrated, by means of a special 
technique, their study was first taken up by the zoologists, as has been shown, 
and special functions in the cell metabolism were imputed to them by 
Meves and others. Pensa is credited with having made the first observa- 
tions tending to show that the mitochondria of plants may, possibly, be 
transformed plastids. This idea, developed by Lewitsky and Pensa and 
supported by numerous observations which have already been noted, was 
taken up by Guilliermond, who has attempted particularly to harmonize 
the functions of the mi ochondria of plant cells with the theories concerning 
those of the mitochondria of animal cells as postulated by Meves, Dues- 
berg, Regaud, Dubreuil, and others. He has confirmed the observations 
of Lewitsky and of Pensa by work upon a number of plants, including the 
seedling of barley. Here the mitochondria, followed from the meristem 
toward the green tip of the plumule, are shown as filamentous at first, 
followed by shorter and thicker forms which are sometimes dumb-bell 
shaped. From the appearance in succeeding cells of bodies which have the 
appearance of the separated halves of the " dumb-bells/' he believes that 
the latter divide. These bodies are followed by more rounded forms with 
a light center and a darker border. Finally, in the tip of the plumule, the 
mature chloroplasts are seen. While this series is considered by Guillier- 
mond a very convincing proof of the mitochondrial origin of the chloroplasts, 
it is open to the objection that there seems to be no way of demonstrating 
that the Anlagen of the plastids are actually mitochondria and not merely 
young plastids. 

On the other hand, the attempts of Rudolph, Sapehin, Mottier, and 



A STUDY OF PLASTIDS AND MITOCHONDRIA 23 1 

others to substantiate their contention that the Anlagen of the plastids are 
different from the mitochondria fail for the same reason — they are unable 
to demonstrate such a difference. Guilliermond ('14) claims that he is, in 
reality, upholding the Schimper-Meyer theory in that he is bridging a gap 
which the latter, with their cruder technique, were unable to fill. Schmidt 
('12) also maintains that the work of Guilliermond and others confirms the 
Schimper-Meyer theory, but for a very different reason : they have simply 
been demonstrating the earlier stages of the plastids, according to Schmidt. 
Lewitsky, however, claims to have shown that there are no other Anlagen 
of the chloroplasts than the mitochondria. His findings, with respect to 
Asparagus officinalis, which have already been referred to, are specifically 
as follows: a stem-tip of Asparagus officinalis was fixed with alcohol, 3 
parts, and acetic acid, 1 part. This was stained with iron-alum-haema- 
toxylin and light green. In the third and fourth cell-layers from above, 
where, in the preparations fixed by the Benda method, the somewhat large 
" chroma tophore-dumb-bells ,, were found, these were no longer to be seen; 
only the usual " Plasmagervst" was present. Since the mitochondria are 
destroyed by acetic acid and alcohol, and since all of the Anlagen, including 
the dumb-bell forms, disappear with the mi ochondria under fixation with a 
combination of the above named reagents, these facts are taken as conclusive 
evidence of the identity of the mitochondria with the Anlagen of the plastids. 

Conclusions 
My own observations may be briefly summarized as follows : 

1 . As to size, an unbroken series of bodies, from mitochondria to plastids, 
can be traced in the root-tip cells of Zea Mays from the embryonic region 
backward. In Preissia this seriation is not so obvious. 

2. The contention that such definitely staining bodies as the mitochon- 
dria exist and are normal constituents of the cytoplasm can hardly be 
questioned. 

3. The evidence for the division of the mitochondria as well as that for 
their functions in heredity seems to me to be inadequate. 

4. The further fundamental question as to the relation of the mitochon- 
dria to the remainder of the cytoplasm and the nature of the material in 
which they are imbedded, has not been cleared up. 

5. Red-staining bodies are present in the plastids of corn, and, in some 
cases, in those of Preissia also. 

BIBLIOGRAPHY 
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Bull. 33: 196-228. 



232 W. C. TWISS 

Davis, B. M. 1899. The spore mother cell of Anthoceros. Bot. Gaz. 28: 89-109. 
Derschau, M. v. 1907. Uber Analogieen pflanzlicher und tierischer Zellstrukturen. 

Beih. Bot. Centralbl. 22: 167-190. 
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7: 424-446. 
D obeli, C. C. 1909. Chromidia and the binuclearity hypothesis. Quart. Journ. Micr. 

Sci. 53: 
Dubreuil, G. 1913. Le chondriome et le dispositif de l'activite secretaire. Arch. Anat. 

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cellules vegetales. Anat. Anz. 36: 96-100. 
Faure'-Fremiet, E. 1908. La structure des matieres vivantes. Bull. Soc. Zool. France 

33: 104-106. 
. 1910. Etude sur les mitochondries des Protozoaires et des cellules sexuelles. 11: 

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Bot. Ges. 29: 648-660. 
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Acad. Sci. (Paris) 153: 199-201. 
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A STUDY OF PLASTIDS AND MITOCHONDRIA 233 

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EXPLANATION OF PLATES XXXIII-XXXIV 

All figures were drawn with the aid of the camera lucida, with a Leitz No. 3 ocular and 
Leitz 1/16 in. oil immersion lens, with the following exceptions: In figure 7 a Leitz No. 6 
objective was used; in figures 13 and 14, a Spencer 1/12 in. oil immersion lens. 

Plate XXXIII 

Fig. I. Cell from plerome of root-tip of corn, showing mitochondria. X 900. 

Fig. 2. Cell from periblem of same preparation, showing mitotic figure and ovoid 
and spherical mitochondria. X 900. 

Fig. 3. Cell from same region showing similar structures. X 1800. All of above 
from Regaud 's fixation and haematoxylin stain. 

Fig. 4. Cell from plerome of corn root-tip, from plant grown under more favorable 
conditions. Benda's method of fixation and staining. Vesicular structures show light 
blue interior. X 900. 

Fig. 5. Two cells from periblem of same preparation. Mitochondria ovoid or spher- 
ical. X 900. 

Fig. 6. Series of plastids from same preparation, stained with Flemming's tri-color 
process. First six in series have red-staining granular inclusions; g, h, and i are stained 
blue. Iodine test shows starch grains in latter. (Fig. 6 on Plate XXXIV.) 

Fig. 7. Section from young disc of Preissia, showing distribution of the granular 
material in the cells and in the thallus as a whole. X 385* 

Figs. 8, 9, and 10. Cells taken from same section, showing plastids, etc., on larger 
scale. X 900. 



234 w - c - twiss 

Plate XXXIV 

Fig. i I . Cell from periphery of young disc of Preissia. Benda's method (unbleached) . 
Large bodies with dark borders, violet; smaller, uniformly-colored structures, brown. 
X 1800. 

Fig. 12. Cell from same region, showing only larger bodies. X 1800. 

Fig. 13. Portion of older disc, showing development of plastids, with large grains of 
storage starch. Iodine stain, with Benda's fixation. X 800. 

Fig. 14. Two cells from central part of same disc, showing fully developed grains. 
X 800. 

Fig. 15. Series of developing starch grains, from near the periphery of the disc to its 
center. X 900. (Fig. 15 on Plate XXXIII.) 

Fig. 16. Cell from thallus of Preissia. Benda's method (unbleached). Large 
"chloro-leucoplasts" light blue, with dark violet peripheries and filled with starch grains. 
Smaller bodies, dark brown. X 900. 



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